Current cloning methods include sequence homology methods such as isothermal assembly (Gibson, D. G. et al., Nature methods 6, 343-345 (2009)), recombination (Walhout, A. J. et al., Science 287, 116-122 (2000)) or design of sequence signatures left by restriction digestion followed by ligation of DNA, such as BioBricks (described in 2003 by Knight, T. from the MIT Artificial Intelligence Laboratory) and GoldenGate (Engler, C., et al., PloS one 3, e3647 (2008)) (for a review, see, DePaoli, H. C., et al., Journal of experimental botany 65, 3381-3393 (2014)). Each method has its disadvantages, and so far, a platform capable of uniting flexibility, fidelity, efficiency and universality for unbiased handling of multiple DNA segments has yet to be developed. The homology-based methods require sequence overlap, which limit the type and order of fragment cloning. Some strategies, such as designing adaptors that allow for sequences to be part of alternate libraries, only partially surpasses this limitation and in the process create scars and intermediary products are often incompatible with future assembling units (Guye, P., et al., Nucleic acids research 41, e156 (2013)). Moreover, PCR-based methods are error prone, and restriction enzyme-based methods require specific recognition sequences to be present at specific sites which will in turn limit the number of fragments based on the number of restriction sites that can be used (Litcofsky, K. D., et al., Nature methods 9, 1077-1080 (2012); Knight (2003).
One way to overcome such limitation is to use restriction enzymes that recognize a sequence outside the fragment of interest (FOI). If two sets of such enzymes are used in an alternating pattern, the same enzymes can be reused forming a ‘cloning loop’. The most recent use of such principles was revealed in the GoldenBraid (GB) method, which introduced the term endless assembly (Sarrion-Perdigones, A. et al., Plant physiology 162, 1618-1631 (2013), Sarrion-Perdigones, A. et al., PloS one 6, e21622 (2011)). Upon creation of different gene collections, carrying a user-defined 4 nucleotide signature, the GB method provides an alternative to homology-based methods by building some transcriptional units and joining them together in vitro. However, the GB method requires multiple libraries, uses linkers/adaptors to produce functional parts, involves software to assist the construct design (Hillson, N. J., et al., ACS synthetic biology 1, 14-21 (2012)) and leaves non-standard signatures, making it difficult to establish a common platform for different laboratories.
In addition to the above problems, restriction enzyme-based methods often obligate a mutation step to be performed within the FOIs at the enzyme recognition sequence in order to properly manipulate the DNA segment, a process called domestication. The prescribed need to use overlap from homology-based methods and the domestication from restriction enzymes-based methods strongly restricts or even excludes several FOI (for example, regulatory regions) in multigene assemblies. Therefore, to properly support synthetic biology and genetic circuit engineering, within the framework of screening and analyzing many alternative and sharable network designs experimentally, these hurdles at the cloning level must be overcome.